A dual-targeted drug inhibits cardiac ryanodine receptor Ca2+ leak but activates SERCA2a Ca2+ uptake

Our study identified a first multi-targeted compound (GM1869) inhibiting RyR2-mediated Ca2+ leak together with activating SERCA2a function that may represent a critical breakthrough for treatment of diastolic Ca2+ leak in heart failure.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): The present manuscript of Wegener et al utilises a number of techniques to identify and test a number of compounds against Ryanodine Receptor leak and SR calcium uptake via SERCA.As a number of arrhythmogenic phenotypes are due to disturbances in calcium handling by these mechanisms, it is therefore of particular interest as compounds affecting these pathways could ultimately be novel antiarrhythmic agents.In general the work has been carried out to a good standard, and the experiments have been well described.I do however, have a number of minor comments 1) In the introduction when CaMKii is being described, there should potentially be some mention of the EPAC pathways that also may contribute to SR leak.
2) At the end of page 4, the BSol domain is mentioned.I believe there needs to be a bit more information about this domain, as this is mentioned without any discussion.
3) When Istaroxime is mentioned, it should also be mentioned that this compound also has additionally mechanisms of action (eg inhibition of Na/K pump) 4) In the first paragraph of the results the term 'least variably' should be replaced by 'most consistently'.5) Spelling error of 'Catecholamine' on page 11. 6) When i look at Figure 4, it appears the CaT decline for the novel compound could be biphasic.Also this representative example shows a slowing of the decline , just like is seen for dantrolene, whereas the majority of the data shows a faster decline.Is this just down to the choice of the 'representative' example, or is it to do with where you took the beginning of the decline from.Would it be worth trying to fit this to a biphasic curve?
Reviewer #2 (Comments to the Authors (Required)): Summary Heart failure and subsequent ca-mishandling is a major source of morbidity and mortality globally.The authors explored 1,4benzothiazepine derivatives purported to combine activation of SR Ca2+ uptake with reduction of Ca2+ spark activity in cardiomyocytes.GM1869 was identified in creased ER Ca signal in HEK293 cells expressing RyR2 and HL-1 cells.GM1869 decreased Ca2+ spark frequency and slightly decreased FDHM in permeabilized cardiomyocytes.GM1869 reduced spark frequency and accelerated Ca re-uptake in RyR2R2474S cells and human hiPSC-CMs.Overall, the authors provide compelling evidence from multiple systems that GM1869 could improve Ca2+ handling in vivo for the treatment of heart failure.These data could be further supported by direct evidence of drug-target interactions.

Major Comments
The affinity of GM1869 is fairly low for clinical application, no?What is with the images in Fig. 4C?Sample images of waves are not particularly clean.Also, the sample traces do not particularly reflect the data reported.Evidence of direct drug binding at it's proposed targets, perhaps from a competition assay, would increases the impact of the manuscript.

Minor comments
The introduction is overly detailed and difficult to follow.It reads as more of a review of Ca handling rather than framing the specific question being addressed in the manuscript.A similar comment could be made regarding the discussion.Manuscript readability would improve from more concise writing.The present manuscript of Wegener et al utilises a number of techniques to identify and test a number of compounds against Ryanodine Receptor leak and SR calcium uptake via SERCA.As a number of arrhythmogenic phenotypes are due to disturbances in calcium handling by these mechanisms, it is therefore of particular interest as compounds affecting these pathways could ultimately be novel antiarrhythmic agents.
In general the work has been carried out to a good standard, and the experiments have been well described.
I do however, have a number of minor comments 1) In the introduction when CaMKii is being described, there should potentially be some mention of the EPAC pathways that also may contribute to SR leak.

Response to reviewer
The authors appreciate the reviewer's comment.The EPAC/NOS1 pathway described by (Pereira et al, 2017) has been included in the Introduction.
(p4: "RyR2 hyper-phosphorylation by Ca2+-calmodulin-dependent kinase (CaMKII) involving exchange protein directly activated by cAMP (Epac2) and nitric oxide synthase 1 (NOS1) (Pereira et al, 2017;Sag et al, 2009;Wehrens et al, 2004),") 2) At the end of page 4, the BSol domain is mentioned.I believe there needs to be a bit more information about this domain, as this is mentioned without any discussion.

Response to reviewer
The authors appreciate the reviewer's comment.The 4 th paragraph at the end of page 4 mentioning the BSol domain has been moved to the Discussion according to the request of Reviewer 2 to shorten the Introduction.The BSol domain is explained there.Thank you for submitting your revised manuscript entitled "A dual-targeted drug inhibits cardiac Ryanodine Receptor Ca2+-leak but activates SERCA2a Ca2+-uptake".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Along with points mentioned below, please tend to the following: -please add ORCID ID for the corresponding author--you should have received instructions on how to do so -please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system -please note that the titles in the system and on the manuscript file must match -the full name (first name, middle name as initials, last name) of each author should be given on the title page -please mark the corresponding Authors on the manuscript title page -please consult our manuscript preparation guidelines https://www.life-science-alliance.org/manuscript-prep and make sure your manuscript sections are in the correct order LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.orgTo upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

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p13: "In detail, ARM210 reversed the destabilization of the bridging solenoid domain (BSol, (Hadiatullah et al, 2022)) of RyR2 induced by the CPVT-associated RyR2-R2474S mutation thereby stabilizing the closed channel conformation (Miotto et al., 2022).")3)When Istaroxime is mentioned, it should also be mentioned that this compound also has additionally mechanisms of action (eg inhibition of Na/K pump) and S4E-H to your main manuscript text -the References in the Supplemental Material file should instead be incorporated into the main Reference list If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
you to revise the figure legend for figure S2 such that the figure panels are introduced in an alphabetical order -please add callouts for Figures S2A-I and S4E-H to your main manuscript text -the References in the Supplemental Material file should instead be incorporated into the main Reference list If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.Thank you for submitting your Research Article entitled "A dual-targeted drug inhibits cardiac Ryanodine Receptor Ca2+-leak but activates SERCA2a Ca2+-uptake".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.